The present invention is in the field of genetic analysis for medical diagnosis, genetic variation research, or genetic engineering. More specifically, the present invention is in the field of nucleic acid analysis.
For many studies involving microarrays, labeled cDNA is often used as a target. This cDNA can be synthesized through either oligo d(T) primers which bind to the poly (A) tail in eukaryotic mRNA or through random primers, in which the actual binding sequences are not known. It is known that during in vitro reverse transcription of RNA, not only the first-strand cDNA is synthesized but also the second-strand cDNA, as reverse transcriptase can use either RNA or DNA as a template (see, e.g., Gubler, 1987. Second-strand cDNA synthesis: classical method. Methods Enzymol. 152:325-9; Gubler, 1987. Second-strand cDNA synthesis: mRNA fragments as primers. Methods Enzymol. 152:330-5; Kim et al., 1996. Human immunodeficiency virus reverse transcriptase. Functional mutants obtained by random mutagenesis coupled with genetic selection in Escherichia coli. J Biol Chem. 271(9):4872-8; Krug, M. S., and S. L. Berger, 1987. First-strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 152:316-25). There may be many mechanisms by which this second-strand priming occurs. Two possible mechanisms have been studied, either the second strand cDNA is synthesized through re-priming of random hexamers with first strand cDNA or through the hairpin loop formation at the 5xe2x80x3 end of first-strand cDNA.
High-density oligonucleotides have been widely used for gene expression analysis. In addition, it is an ideal platform for other applications like transcriptome analysis, antisense detection, splice variant detection, genotyping, etc. Some of these applications use random hexamer cDNA synthesis for target preparation. The synthesis of second strand cDNA would make the data analysis complicated due to the additional strand synthesis (e.g., antisense RNA could not be identified). Therefore, there is a need in the art for methods that can uniquely identify the sense strand. In addition, methods for identifying the template strand of a genomic DNA are needed.
In one aspect of the invention, methods are provided for detecting a plurality of transcripts without the interference of second strand DNA. The methods include synthesizing a plurality of cDNAs complementary with the transcripts by reverse transcription; where the synthesis of second strand cDNA is inhibited; and hybridizing the cDNAs or nucleic acids derived from the cDNAs with a nucleic acid probe array to detect and identify the transcripts. The methods are particularly suitable for detecting a large number of, at least 100, 1000, or 10000, transcripts. Any suitable second strand cDNA synthesis inhibition methods are suitable for use with at least some embodiments of the invention. In particularly preferred embodiments, hairpin loop formation inhibition is used to inhibit second strand cDNA synthesis. In one particularly preferred embodiment, the synthesis of the second strand cDNA is inhibited by the presence of actinomycin D, DMSO or sodium pyrophosphate. The cDNAs or nucleic acids derived from the cDNAs (e.g., products of PCR amplification of the cDNAs, etc.) may be labeled with any suitable labels, such as radioactive labels, fluorescent labels, and chemoluminescent labels, etc.
The nucleic acid array can be a high density oligonucleotide probe array with at least 400, 1000, 10000 probes per cm2. In preferred embodiments, the array contains at least one probe against a target sequence and one probe against the reverse complementary sequence of the target sequence. In more preferred embodiments, the array contains at least 100 probes against at least 100 target sequences and at least 100 probes against at least 100 reverse complementary sequences of the target sequences. In even more preferred embodiments, the array comprises at least 1000 or 3000 probes against at least 1000 or 3000 target sequences and at least 1000 or 3000 probes against at least 1000 or 3000 reverse complementary sequences of the target sequences.
In another aspect of the invention, methods are provided for detecting the transcribed regions of a genome and the template strand of the genomic DNA. The methods are particularly suitable for analyzing regions where both strands of the genomic DNA may be transcribed. In preferred embodiments, the methods include obtaining a sample containing transcripts transcribed from the genome; synthesizing single stranded cDNAs complementary with the transcripts, where the synthesis of second strand cDNA is inhibited; and hybridizing the cDNAs or nucleic acids derived from the cDNAs with a nucleic acid probe array, where the nucleic acid probe array has probes targeting both strands of the genomic DNA in interested regions. Any suitable second strand cDNA synthesis inhibition methods are suitable for use with at least some embodiments of the invention. In particularly preferred embodiments, hairpin loop formation inhibition is used to inhibit second strand cDNA synthesis. In one particularly preferred embodiment, the synthesis of the second strand cDNA is inhibited by the presence of actinomycin D. The cDNAs or nucleic acids derived from the cDNAs (e.g., products of PCR amplification of the cDNAs, etc.) may be labeled with any suitable labels, such as radioactive labels, fluorescent labels, and chemoluminescent labels, etc. The nucleic acid array can be a high density oligonucleotide probe array with at least 400, 1000, 10000 probes per cm2. In preferred embodiments, the array contains at least one probe against a target sequence and one probe against the reverse complementary sequence of the target sequence. In more preferred embodiments, the array contains at least 100 probes against at least 100 target sequences and at least 100 probes against at least 100 reverse complementary sequences of the target sequences. In even more preferred embodiments, the array comprises at least 1000 or 3000 probes against at least 1000 or 3000 target sequences and at least 1000 or 3000 probes against at least 1000 or 3000 reverse complementary sequences of the target sequences.
In yet another aspect of the invention, an assay kit is provided. The kit contains reagents necessary for a reverse transcription reaction; an inhibitor of second strand cDNA synthesis; and a nucleic acid probe array. In preferred embodiments, the inhibitor is actinomycin D. The nucleic acid probe array is an oligonucleotide probe array that has at least 400, 1000, 10000 probes per cm2.